Chronic pain disorders are often associated with negative emotions, including anxiety and depression. The central nucleus of the amygdala (CeA) has emerged as an integrative hub for nociceptive and affective components during central pain development. Prior adverse injuries are precipitating factors thought to transform nociceptors into a primed state for chronic pain. However, the cellular basis underlying the primed state and the subsequent development of chronic pain remains unknown. Here, we investigated the cellular and synaptic alterations of the CeA in a mouse model of chronic muscle pain. In these mice, local infusion of pregabalin, a clinically approved drug for fibromyalgia and other chronic pain disorders, into the CeA or chemogenetic inactivation of the somatostatin-expressing CeA (CeA-SST) neurons during the priming phase prevented the chronification of pain. Further, electrophysiological recording revealed that the CeA-SST neurons had increased excitatory synaptic drive and enhanced neuronal excitability in the chronic pain states. Finally, either chemogenetic inactivation of the CeA-SST neurons or pharmacological suppression of the nociceptive afferents from the brainstem to the CeA-SST neurons alleviated chronic pain and anxio-depressive symptoms. These data raise the possibility of targeting treatments to CeA-SST neurons to prevent central pain sensitization. Prior adverse events or perceived physical injuries are one of the precipitating factors for chronic pain . A priorCentral sensitization is believed to underlie widespread MP and affective symptoms in the fibromyalgia syndrome . HoweverHere, we aimed to investigate the cellular basis underlying MP using the acid-induced MP model. By combining electrophysiology, chemogenetics, and in vivo calcium imaging, we attempted to demonstrate that CeA-SST neurons, which are believed to be antinociceptive neurons, become hyperexcitable following MP development. Consistent with this finding, selective chemogenetic inactivation of CeA-SST neurons or suppression of synaptic transmission of the PBN to the CeA-SST neurons alleviated MP and negative emotions.Acid-induced MP in rats or mice is considered a preclinical model of fibromyalgia . In thisIn addition to mechanical allodynia, the mice with MP showed various affective symptoms . The eleAnxiety disorders are often associated with depression-like behavior and social avoidance . The forTherefore, we tested the predictive validity of this model. Pregabalin (PGB) is a Food and Drug Administration-approved drug for the treatment of chronic pain disorders and affective comorbidities. PGB is a selective ligand for the α2δ subunit of voltage-gated calcium channels VGCCs; . HoweverIn this model, the first acidic saline injection initiated a priming phase, which reportedly lasts for 5–8 days . TherefoTo test whether local PGB administration in the CeA also alleviated negative emotions in the MP mice, we infused PGB into the CeA of the Ctrl and MP mice bilaterally through a cannula . CannulaConsidering that the PGB treatment during the priming phase is sufficient for preventing chronic pain development, we also tested whether PGB delivery during the priming phase can prevent comorbid affective symptoms. Thus, we infused ACSF or PGB into the CeA of the MP mice bilaterally through a cannula right after the second acidic saline injection . The samTo characterize maladaptive changes in the amygdala circuits in chronic MP states, we recorded the excitatory synaptic transmission to the CeA-SST and CeA-PKCδ neurons using whole-cell patch-clamp recording in brain slices from the Ctrl and MP mice . Using tNext, we investigated whether the intrinsic excitability of the CeA-SST and CeA-PKCδ neurons was altered in the MP mice . To deteThe increased CeA-SST excitability was associated with chronic pain phenotypes in the MP mice. We sought to determine the causal role of the CeA-SST neurons in the MP development. Therefore, we tested whether suppression of neuronal excitability of the CeA-SST neurons during the initial muscle injuries was sufficient to interfere with the development of chronic pain states. A Cre-dependent adeno-associated virus serotype 5 (AAV5) encoding an inhibitory designer receptor (hM4Di) was injected bilaterally into the CeA of SST-Cre mice . Most ~. Whole-cNext, we tested the causal role of the CeA-SST neurons in MP-associated behaviors. CNO was i.p. injected into the mice following the establishment of MP . We founBoth enhanced CeA-SST and decreased CeA-PKCδ neuronal activity were observed in the MP mice. These two types of neurons reciprocally inhibit each other. We subsequently tested whether increasing CeA-PKCδ neuronal activity could also reduce mechanical hyperalgesia and anxiety-like behaviors. The excitatory designer receptor (hM3Dq) was virally expressed in the CeA-PKCδ neurons . Most ofSince enhanced CeA-SST neuronal activity was observed in the MP mice, we tested the effect of PGB on excitatory synaptic transmission onto the CeA-SST neurons. To this end, we injected an AAV5-CaMKIIα-ChR2-eYFP virus into the PBN of the SST-Cre;Ai14 mice. Four weeks later, these mice were randomly included as either the Ctrl or MP mice . Using tFinally, we tested whether intraperitoneal injection of PGB suppresses the CeA-SST neuron activity using in vivo calcium imaging. To selectively detect the calcium activities of the CeA-SST neurons, the SST-Cre mice were virally transduced to express the calcium indicator GCaMP6s and implanted with the GRIN lens above the CeA for 4 weeks before the test. Calcium activities of single CeA-SST neurons were detected before and 45 min following intraperitoneal injection of PGB , SST-Cre;Ai14 (SST-Cre line crossed with Ai14 line), PKCδ-Cre (stock no. 011559), and PKCδ-Cre;Ai14 (PKCδ-Cre line crossed with Ai14 line). The SST-Cre line and Ai14 tdTomato reporter (stock no. 007914) were purchased from the Jackson Laboratory. PKCδ-Cre and C57BL/6J mice were purchased from the Mutant Mouse Resource and Research Centers (MMRRC) and National Laboratory Animal Center, respectively. Mice aged 2–5 months of either sex were used in the electrophysiological and behavioral studies. All the mice were bred against the C57BL/6J genetic background. Mice were housed in a 12 hr light-dark cycle and provided food and water ad libitum. Two-month-old mice were injected with a virus and implanted with optical fibers in the CeA. The animals were handled in accordance with national and institutional guidelines. All behavioral procedures were conducted in accordance with the protocol (no. 1080317) approved by the Institutional Animal Care and Use Committee of the National Yang Ming Chiao Tung University.To specifically express designer receptors exclusively activated by designer drugs (DREADDs) onto the CeA-SST and CeA-PKCδ neurons, we used a recombinant AAV5 carrying hM3Dq or hM4Di conjugated to mCherry in a double-floxed inverted open reading frame (DIO), driven by the human Synapsin I (hSyn) promoter (AAV5-hSyn-DIO-hM3Dq-mCherry or AAV5-hSyn-DIO-hM4Di-mCherry). In the optogenetic experiments, AAV5 carrying channelrhodopsin-2 (ChR2) conjugated to eYFP driven by the CaMKIIα promoter (AAV5-CaMKIIα-ChR2-eYFP) was used to selectively express ChR2 onto the PBN neurons. To silence the neurons, AAV5-hSyn-DIO-hM4Di-mCherry was injected into the CeA region. To activate the neurons, the AAV5-hSyn-DIO-hM3Dq-mCherry virus was injected into the CeA region and the AAV5-CaMKIIα-ChR2-eYFP virus was injected into the PBN region. In addition, a viral vector carrying the red fluorescent protein (AAV5-hSyn-DIO-mCherry) was used as the control. In the in vivo calcium imaging experiment, the AAV5-Syn-Flex-GCaMP6s-WPRE-SV40 was injected into the CeA region. All viral vectors were purchased from the Vector Core at the University of North Carolina  or Addgene Vector Core .2; Halocarbon Laboratories, North Augusta, SC, USA). After anesthesia, the MP and Ctrl mice received 20 μL injections of acidic saline (pH 4.0) and neutral saline (pH 7.2), respectively, on day 0 in the left gastrocnemius muscle. After 3 days (day 3), the same gastrocnemius muscle was re-injected with acidic or neutral saline. The pH value of the 2-(N-morpholino) ethanesulfonic acid (MES)-buffered saline  was used to construct both the acidic and neutral saline, while the pH values were adjusted with 0.1 M of HCl or NaOH.The model of acid-induced MP  was used2O2 for 10 min and then blocked with 0.1% Triton X-100 in TBS containing 2% bovine serum albumin and 2% normal goat serum  for 2 hr at room temperature. To confirm the expression of the virus in the CeA, the slices were stained with a primary antibody: rabbit anti-red fluorescent protein  or rabbit anti-PKCδ  or rat anti-SST . After 2 days, the slices were stained with a secondary antibody: goat anti-rabbit Alexa Fluor 594  or goat anti-rabbit Alexa Fluor 488  or goat anti-rat Alexa Fluor 488 . The results were examined and imaged under a fluorescence microscope  or a confocal laser excitation microscope .Mice were deeply anesthetized and PBS  was sequentially perfused through the left ventricle, followed by 30 mL of ice-cold 4% paraformaldehyde in 0.1 M of PBS. The brain was rapidly removed and fixed in 4% paraformaldehyde in 0.1 M of PBS for 6 hr at 4°C, after which it was cryoprotected with 15% sucrose in 0.1 M of PBS for 24 hr at 4°C, followed by 30% sucrose in 0.1 M of PBS for 24 hr at 4°C. Coronal brain sections (45 μm thickness) containing the amygdala and surrounding regions were cut using a cryostat microtome . Sections were treated with 3% H2; Halocarbon Laboratories, North Augusta, SC, USA) and placed in a stereotaxic injection frame . The injections were performed using the following stereotaxic coordinates: for the CeA, 1.31 mm posterior from bregma, 2.87 mm lateral from the midline on both sides, and 4.72 mm ventral from the cortical surface; for the PBN, 5.1 mm posterior from bregma, 1.2 mm lateral from the midline on both sides, and 3.2 mm ventral from the cortical surface. During all the surgical procedures, the mice were kept on a heating pad  to maintain their surface body temperatures at 34°C. After securing the head with ear bars, 75% ethanol was used to sterilize the surgical area, and the eyes were protected using an ophthalmic gel. For viral injection, we injected 0.35 and 0.5 μL of the viral solution bilaterally into the CeA and PBN, respectively, using a 10 μL NanoFil syringe and a 34-gauge (G) beveled metal needle . The flow rate (0.1 μL/min) was controlled with a nanopump controller . After viral injection, the needle was raised 0.1 mm above the injection site for an additional 10 min to allow the virus to diffuse before being withdrawn slowly. To reach optimal viral expression, all the animals were allowed to recover for at least 4 weeks before conducting behavioral and electrophysiological experiments.Mice were deeply anesthetized with isoflurane  and dummy  cannulas. The guide cannulas were placed in the CeA bilaterally . To fix the guide cannulas onto the skull, dental resin cement C&B Super-Bond  was applied to the surface of the skull around the cannula for approximately 10 min. After the resin cement hardened, the cannula was removed from the homemade holder and the mice were placed back into their home cages for recovery. One week after recovery, the mice underwent behavioral tests. During the test, the dummy cannulas were replaced by internal cannulas . We bilaterally injected 0.15 μL of ACSF at a rate of 75 nL/min through the cannulas on days 15 and 18. For the delivery of PGB , we bilaterally injected 0.15 μL of 1 mM PGB at a rate of 75 nL/min through the cannulas on days 16 and 19. For the priming experiment, we bilaterally injected 0.15 μL of ACSF or PGB through the cannulas right after the second acidic saline injection on day 3.After injection of the virus, the GRIN lens  was placed in the CeA  region. To fix the lens onto the skull, dental resin cement C&B Super-Bond  was applied to the surface of the skull around the lens for approximately 10 min. After the resin cement hardened, the lens was removed from the holder and the mice were placed back into their home cages for recovery. Three to four weeks after injection of the virus, the mice underwent MP induction. Two weeks after MP induction, the calcium activities of the CeA-SST neurons were recorded. Calcium signals were detected using the miniscope system . The blue light-emitting diode (LED) power was approximately 1.4 mW at the focal plane. The images (1280×800 pixels) were acquired at a 15 or 20 Hz frame rate. After adapting the miniscope, the calcium imaging was recorded from freely moving mice in the home cage. The analysis was performed using the Inscopix data processing software . The constrained nonnegative matrix factorization was applied to extract calcium event traces (ΔF/F) of all the recorded CeA-SST neurons.Before and after the PGB treatment, the frequencies and mean amplitudes of the events were detected using the built-in template searching function . For the AUC analysis, in order to eliminate calcium-independent residual noise and to improve the signal-to-noise ratio, the Gaussian kernel filter  sucrose saline-containing chamber at 34°C for 30 min before being maintained at room temperature until recording. During the experiment, slices were transferred to a submerged chamber and perfused with oxygenated ACSF containing the following (in mM): 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 25 glucose, 2 CaCl2, and 1 MgCl2. The expression of the virus or tdTomato expression was confirmed by red or green fluorescence and the neurons in the CeA were visually selected for recordings under an infrared differential interference contrast microscope  equipped with an LED source . For optical stimulation, ChR2-expressing neurons were excited by 470 nm LED light . Whole-cell patch-clamp recordings were conducted using an Axopatch 200B amplifier . Recording electrodes (3–6 MΩ) were pulled from borosilicate glass capillaries . The glass microelectrodes were filled with a low Cl- internal solution containing the following (in mM): 136.8 K-gluconate, 7.2 KCl, 0.2 EGTA, 4 MgATP, 10 HEPES, 7 NA2-phosphocreatine, 0.5 Na3GTP, and 0.4% biocytin . The pipette capacitance was compensated. Signals were low-pass filtered at 5 kHz and sampled at 10 kHz using a digitizer . In the ex vivo slice recordings, the following antagonists were added to the ACSF: SR-95531 , CGP-55845 , and kynurenic acid  to block the GABAA, GABAB, AMPA/NMDA receptors, respectively. Tetrodotoxin  was applied to block sodium channels in the experiments of the mEPSC recording. CNO  was used to activate the DREADDs. PGB  was used to examine its potential effects on synaptic transmission at the PBN to the CeA-SST or CeA-PKCδ neuron synapses.After the behavioral tests, AAV-injected mice were sacrificed and acute coronal brain slices of 300 μm thickness were cut using a vibratome  in ice-cold sucrose saline containing the following (in mM): 87 NaCl, 25 NaHCOMice were handled for at least 3 days before the behavioral tests . All theMechanical hypersensitivity was assessed using the von Frey filament test. A series of von Frey filaments of increasing stiffness (0.04–1.4 g) were applied to the plantar surface of both the hindpaws. Each filament was applied five times and the lowest force that caused at least three withdrawals out of the five stimuli was considered the threshold (g) .The clean cage  was filled approximately 6 cm high with bedding. Twenty-four glass marbles (approximately 1.5 cm in diameter) were evenly spaced on the top of the bedding with an approximately 3 cm distance between each pair of marbles. Mice were placed individually into the cages and left undisturbed for 30 min . MarblesThe test apparatus consisted of a two-compartment light-dark box  connected by a central opening at the floor level. Mice were placed individually in the center of the brightly lit side of the box and left undisturbed for 10 min of exploration. Transitions between the two compartments and total locomotor activity were recorded using the Tru-scan 2.0 system .The EPM is a common anxiety test that produces approach avoidance conflict . The EPMThe three-chamber sociability test has been successfully employed to study social affiliation in several mouse lines . The socThe FST consisted of a transparent acrylic cylindrical container  filled with water to a height of 16 cm . The mouBL. Statistical significance was tested using the two-way analysis of variance (ANOVA) with the Tukey’s post hoc test, two-way ANOVA with the Bonferroni’s multiple comparison test, Kolmogorov–Smirnov test, Wilcoxon matched-pairs signed rank test, or Mann–Whitney test at the significance level (p) indicated. Data are presented as mean ± standard error of mean (SEM). Significance levels were set at p<0.05 (*), p<0.01 (**), p<0.001 (***), or p<0.0001 (****).Data were analyzed using Clampfit 10.3 , MiniAnalysis , GraphPad Prism 6  and a custom-made program written in Python. To define the effect of PGB, we classified PGB-inhibited neurons as having the EPSC inhibition >×1 SD This study investigates the role of the central amygdala (CeA) on the mechanisms underlying chronic pain. An acid-induced muscle pain (AIMP) mouse model is used. Changes of excitatory synaptic and intrinsic excitability of GABAergic somatostatin-expressing (SST+) neurons of the CeA are detected in the chronic MP model. The manuscript is extensive, well-illustrated and contains valuable findings that are likely to inspire future work to progress the understanding of the GABAergic system on patho-physiological events. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Marco Capogna as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Gary Westbrook as the Senior Editor.Thank you for submitting your article "Cellular Mechanisms Underlying Central Sensitization in a Mouse Model of Chronic Muscle Pain" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:eLife is two months, but some more experiments will be required. Thus the authors should take the time required to address the issues below in a convincing manner as well as respond to the comments of the original reviews.The reviewers and editors agreed that this manuscript is interesting, but they also raised criticism that needs to be taken into consideration. We would like to ask you to prepare a revised version of the manuscript and re-submit. The ideal deadline for re-submission for 1) The investigation of the direct monosynaptic excitatory input from the PBN to CeA neurons should be completed by recordings from PKCd+ neurons. Furthermore, more statistical analyses of the data already submitted should be performed, as explained by Reviewer 1.2) Increase the number of animals used with chronic muscle pain treated with PGB for the EPM and sociability behavioral tests.3) Provide control data on post-hoc images of the virus injection/expression and co-localization with cell type specific markers; refer to previously published data or publicly available data (Allen Inst) on a2d VGCC subunit expression in parabrachial -CeA.Reviewer #1 (Recommendations for the authors):4) Better discuss: acute PGB effect on the mechano-sensitivity versus emotional behavior; mechanisms involved in changes in intrinsic excitability of neurons in the MP model; pre- versus pot-synaptic changes detected in the MP model; the source(s) of the spontaneous synaptic events.1) The direct monosynaptic excitatory input from the parabrachial nucleus (PBN), which is enriched with the calcium channel α2δ subunit, onto CeA neurons, is directly investigated by presynaptic optogenetic stimulation and postsynaptic recordings of SST neurons . I think this is an important approach that should be further exploited. From the data submitted, it seems that such evoked EPSCs are about 3 times bigger in MP mice compared to controls. Provided that optogenetically-evoked EPSCs from control and MP mice could be appropriately compared, e.g. through normalization, is this difference statistically significant?It would be also useful to statistically compare the inhibition of the evoked EPSCs by PGB in the two genotypes. Finally, could the Authors perform some more experiments and report the results of a similar experiment by recording from PKCδ neurons, and statistically compare it to the evoked EPSCs recorded in SST neurons?2) What mechanisms the Authors envisage to account for by the changes in intrinsic excitability in SST+ and PKCδ neurons observed in MP mice? This aspect should be discussed.3) The Authors reported effects on amplitude and/or frequency of sEPSCs and mEPSCs onto CeA recorded neurons seem to suggest a presynaptic mechanism of action. In contrast, the effect of PGB on calcium transient revealed by 1P deep calcium imaging may be interpreted as pre and/or postsynaptic.The Authors should make a more definitive conclusion whether they think their data suggest a presynaptic only or also a postsynaptic mechanism of action. It may be difficult to draw any conclusion from sEPSCs since these events are probably due to the release of multiple synaptic vesicles .4) The Authors should discuss the possible source(s) of the spontaneous synaptic events. Do they only originate from PBN terminals or also from BLA principal neurons or elsewhere? Is an analysis of mEPSCs kinetics compatible with an homogeneous pool of presynaptic terminals as source of the m/sEPSCs or rather SST+ and PKCδ+ neurons are biophysically too compact to make out the presence of heterogeneous excitatory inputs perhaps impinging on different spatial domains of the neurons' dendritic trees?5) The paw withdraw effect could be replicated by either reducing the CeA SST+ cell activity or by elevating PKCd+ cell activity, but this is not the case for the anxiety-like affective behaviors . Therefore, the PGB effect on the increased paw withdraw threshold and emotion behaviors in the MP mouse model may be caused by distinct circuit mechanisms occurring simultaneously.To further elucidate their observation, the Author should test the acute PGB effect on the mechano-sensitivity and emotional behavior separately using a wild type mice or should at least acknowledge this possibility in the discussion.Reviewer #2 (Recommendations for the authors):My specific issues are outlined below:1) Why does the number of animals tested in the different behavioural paradigms  vary from test to test? Were these different cohorts for each behavioural test?2) The size of the cohorts in which PGB was tested (and the relative controls) after induction of chronic muscle pain  is suboptimal for the EPM and sociability tests, also in view of the known relatively high interindividual variability, and should, therefore, be increased.2+ imaging experiments were performed in mice with chronic muscle pain. Why did the authors did not test the effect of PGB on SST+ neuron activity in control animals? This would have strengthened their findings. Moreover, they could have better exploited their study by testing the effect of PGB on SST+ neuron activity during a von Frey filament stimulation, as this test offers good behavioral bouts in which to analyse ca2+ transients.3) It should be made more clear in the text that in vivo caReviewer #3 (Recommendations for the authors):I have the following comments, which require more experiments to address in order to make the manuscript more convincing.1, Figure 3 shows recording results from CeA-SST and CeA-PKCd neurons in the MP mice. Since these neurons at baseline consist of different firing phenotypes , it would be useful to know whether all firing types show the same changes in synaptic transmission and excitability in the MP model.Notably, in Wilson et al., 2019 paper, they found that CeA neurons require at least 50-80 pA current injection to induce the spikes, whereas in this manuscript, CeA neurons require a much lower current injection to fire. Could the author provide some explanation to this difference?2, The number of animals in each test is quite different. For instance, in Figure 4E, n=10 for the mCherry group and n=52 for the hM4Di group; this is a big difference to compare 2 groups. Also, in Figure 4 supplement 2, in the CeA-PKCd experiment, n=4 for the mCherry group and n=13 for the hM3q group. These numbers are significantly fewer than those for CeA-SST hM4Di group (n=52). Please explain how the numbers of animals were assigned to each test? Did the author exclude some animals from some tests?3, The authors did not show any post-hoc images of the virus injection/expression and co-localization with cell type specific markers for all the functional manipulation and imaging studies. These should be provided to substantiate the results.4, In Figure 4 supplement 1B: Chemogenetic inhibition of CeA-SST neurons does not affect the paw withdrawal response in the naïve mice. In contrast, the authors themselves showed in their recent paper that optogenetic inhibition of CeA-SST neurons increased paw withdrawal response . Please reconcile these two different results in the same lab.5, There is no data on a2d VGCC subunit expression in parabrachial or CeA in the chronic MP model in this manuscript. Given so much emphasis on PGB results, this information is crucial and is missing here.6, The authors performed the in vivo calcium imaging of CeA-SST neurons in response to PGB. It would be more informative to know how CeA-SST neurons or CeA-PKCd neurons respond to 1st and 2nd acid injection.eLife. Your revised article has been evaluated by Gary Westbrook (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Cellular Mechanisms Underlying Central Sensitization in a Mouse Model of Chronic Muscle Pain" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined here and in the novel comments by the reviewers:Essential revisions:All the reviewers acknowledged that the Authors replied satisfactorily to most of their comments and that the first revised version is improved. However, they also concur that the manuscript still needs some significant improvements.Specifically:1) The text is often hard to read and should be carefully checked to improve readability. If needed, a professional writer could be helpful.2) All the reviewers acknowledged the additional excel database and your explanations regarding the data in Figure 3 – supplement 2 B-D. These data were submitted to support the electrophysiological results – that SST+ neuron excitability is selectively enhanced in MP mice. However, some improvements are needed:a) We think that you pooled the data from "all slices" together at a given AP position relative to Bregma, to generate the plots and calculate SEM. However, we think you should re-calculate the mean at each AP position for each animal first, then average across animals and calculate SEM. In this way, the data would be averaged within an animal and the variance would be calculated between animals in order to assess the robustness of the results;b) Explain why the number of slices at a given AP position varies dramatically across animals . Furthermore, how it could be that in some mice the AP position has 5-9 slices, whereas from the method one can calculate that a maximum of 3 slices can be obtained for a given AP position;c) The number of samples for each bregma level and animal should be similar and more control data should be collected and included since currently they show large variability;d) Statistical analysis should be improved, e.g. a two-factor analysis of variance  would be a good way to analyze these data.Reviewer #1 (Recommendations for the authors):I think that the Authors have done a good job in revising the manuscript according to my suggestions.Specifically:1) They further investigated and analyzed synaptic transmission at the PBN input to the CeA. The data suggest that the PBN input is not the major potentiated input onto the CeA-SST neurons in the MP mice. They also compared the effect of PGB inhibition between the Ctrl and MP groups and found it similar. Then, they tested the effect of PGB on synaptic transmission at the PBN-CeAPKCδ neuron synapses. Approximately 30% CeA-PKCδ neurons were sensitive to PGB inhibition in the Ctrl mice and approximately 63% CeA-PKCδ neurons were sensitive to PGB inhibition in the MP mice.2) Potential changes in intrinsic excitability of CeA-SST neurons are discussed in the revised version of the manuscript.3) The Authors strengthen the interpretation of enhancement of vesicle release at the inputs onto the CeA-SST neuron synapses along with the increased excitability to account for the central sensitization of the CeA-SST neurons in MP.4) They addressed the issue of PGB mediated increase in paw withdraw threshold and emotion behaviors.Reviewer #2 (Recommendations for the authors):The work by Lin and coworkers addresses the important and of broad interest process that underlies the progression of transient pain into persistent pain. This study used a number of sophisticated techniques to dissect the synaptic and cellular mechanisms that occur in the mouse central amygdala (CeA) during the development of chronic muscle pain. In particular, the study reveals shifts in opposite directions of the activity of somatostatin-expressing (SST+) and PKCδ+ neurons. Pharmacological and chemogenetic manipulation of these neurons prevented the chronification of pain and of the comorbid affective behaviors, although with important differences between these two neuronal classes.In their revised manuscript, the authors have made a number of significant changes by which they could adequately address my main concerns. In particular, the additional experimental data aimed at increasing the sample size of the behavioural experiments has appreciably improved the manuscript.While the text has improved, it still contains several syntax and grammar errors that need to be corrected.Reviewer #3 (Recommendations for the authors):In the revised manuscript, the authors addressed most of my previous concerns. In their response to my question of conflicting results for inhibition of CeA-SST neurons from their prior publication, they responded that "It seems that different illumination protocols resulted in opposite behaviors". "5 min optogenetic inhibition of CeA-SST reduced mechanical sensitivity, while 10 min illumination increased mechanical sensitivity". I honestly cannot think of any known mechanism that could explain opposing effect induced by 5min vs 10min optogenetic inhibition. I give up.I do have some questions for Figure 3—figure supplement 2+ cells in B being less than that in D (a subset of neurons)?In D, the number of SST(-)pERK(+) in the Ctrl (-1.82 mm) is above 40, while in B, the number of total pERK(+) in the Ctrl (-1.82mm) is about 30. This is weird. How could total number of pERKAuthors concluded in Line 212-213, "increased pERK-positive cells are mostly CeA-SST cells." From the results shown in C and D, it's difficult to be convinced of this statement: the number of SST(+) pERK(+) cells increased from 1.1 to 3.7, while SST(-) pERK(+) cells increased from 18 to 28, even though statistics appeared to be not significant. I believe that instead of comparing the averaged number across sections, comparing the total number of cells in the CeA makes more sense.+) and in panel D (SST-/pERK+) appear identical. Could there be an image duplication here?The "gray" bars for "control" samples in panel B  The investigation of the direct monosynaptic excitatory input from the PBN to CeA neurons should be completed by recordings from PKCd+ neurons. Furthermore, more statistical analyses of the data already submitted should be performed, as explained by Reviewer 1.In the revised version of the manuscript, we completed the investigation of the direct monosynaptic excitatory input from the PBN to the CeA-PKCδ neurons . As the reviewer #1 suggested, we also performed additional statistical analyses of the data (see our response to the reviewer #1).2) Increase the number of animals used with chronic muscle pain treated with PGB for the EPM and sociability behavioral tests.In the revised version of the manuscript, we increased the number of experiments in Figure 2B  and 2E .3) Provide control data on post-hoc images of the virus injection/expression and co-localization with cell type specific markers; refer to previously published data or publicly available data (Allen Inst) on a2d VGCC subunit expression in parabrachial -CeA.We added images of virus expression (AAV5-hSyn-hM4Di-mCherry or AAV5-hSyn-hM3Dq-mCherry) colocalized with cell type specific markers (SST or PKCδ) in Figure 4B and Figure 4 —figure supplement 4B in the revised submission. The colocalization of specific markers with virus expression was summarized in Figure 4 —figure supplement 1 and 4B in the revised submission.Regarding the α2δ VGCC subunit expression, we cited a previous study by Cole et al. (2005), which demonstrated the anatomical distribution of α2δ VGCC subunit expression across the whole rat brain . Moreover, we provided the published data by Allen Brain Institute, which demonstrated α2δ1 and α2δ2 VGCC subunit expression in the parabrachial nucleus in our supplement file .4) Better discuss: acute PGB effect on the mechano-sensitivity versus emotional behavior; mechanisms involved in changes in intrinsic excitability of neurons in the MP model; pre- versus pot-synaptic changes detected in the MP model; the source(s) of the spontaneous synaptic events.In the revised version of manuscript, we included discussions on acute PGB effect on the mechano-sensitivity versus emotional behavior , mechanisms involved in changes in intrinsic excitability of neurons in the MP model , pre- versus post-synaptic changes detected in the MP model , and the source(s) of the spontaneous synaptic events . Please also refer to our response to the Reviewer #1 (point #1 – 5).Reviewer #1 (Recommendations for the authors):1) The direct monosynaptic excitatory input from the parabrachial nucleus (PBN), which is enriched with the calcium channel α2δ subunit, onto CeA neurons, is directly investigated by presynaptic optogenetic stimulation and postsynaptic recordings of SST neurons . I think this is an important approach that should be further exploited. From the data submitted, it seems that such evoked EPSCs are about 3 times bigger in MP mice compared to controls. Provided that optogenetically-evoked EPSCs from control and MP mice could be appropriately compared, e.g. through normalization, is this difference statistically significant?It would be also useful to statistically compare the inhibition of the evoked EPSCs by PGB in the two genotypes. Finally, could the Authors perform some more experiments and report the results of a similar experiment by recording from PKCδ neurons, and statistically compare it to the evoked EPSCs recorded in SST neurons?p = 0.13; Figure 5 —figure supplement 2A). Moreover, the PPR of the light-evoked EPSCs recorded at the PBN-CeA-SST neuron synapses showed no change in the Ctrl and MP groups . These results may suggest that the PBN input is not the major potentiated input onto the CeA-SST neurons in the MP mice. However, it is worth noting that the CeA-SST neurons were randomly selected for recording. It is very possible that not all recorded CeA-SST neurons were sensitized neurons. Therefore, we need to carefully interpret our data.In revision, we further investigated and analyzed synaptic transmission at the PBN input to the CeA. First, we found the average EPSC amplitude was 1.5 times greater in the MP mice compared to the Ctrl mice. However, the change did not reach the statistical significance  and the degree of inhibition between these two groups was similar .Second, we compared the effect of PGB inhibition between the Ctrl and MP groups . Bath application of PGB (500 µM) decreased the amplitude of light-evoked EPSCs in some CeA-SST neurons of both the Ctrl and MP mice . Overall, the percentage of PGB-sensitive neurons was only slightly increased in the MP mice . However, the paired-pulse ratio of the light-evoked EPSCs recorded at the PBN-CeA-PKCδ neuron synapses was not different between the Ctrl and MP groups . Compared to the amplitude of the light-evoked EPSCs at the PBN-CeA-SST neuron synapses, the light-evoked EPSCs amplitude of the CeA-PKCδ neurons was increased in the Ctrl mice , but showed no significant change in the MP mice . In addition, the paired-pulse ratio of the light-evoked EPSCs recorded at the synapses of the PBN-CeA-PKCδ neurons and PBN-CeA-SST neurons were not different between the Ctrl  and MP groups . Bath application of PGB decreased the amplitude of light-evoked EPSCs in the CeA-PKCδ neurons of both the Ctrl and MP mice . Approximately 30% CeA-PKCδ neurons were sensitive to PGB inhibition in the Ctrl mice and approximately 63% CeA-PKCδ neurons were sensitive to PGB inhibition in the MP mice . Furthermore, the degree of inhibition between these two groups were similar . Consistent with the effect of PGB on presynaptic α2δ subunit of VGCCs, the reduction of EPSCs was concomitant with an increase in the paired-pulse ratio .Finally, we tested the effect of PGB on synaptic transmission at the PBN-CeA-PKCδ neuron synapses. The amplitude of the light-evoked EPSCs recorded at the PBN-CeA-PKCδ neuron synapses was decreased in the MP group  What mechanisms the Authors envisage to account for by the changes in intrinsic excitability in SST+ and PKCδ neurons observed in MP mice? This aspect should be discussed.Several molecular mechanisms underlying aberrant plasticity of sensitized neurons were reported in different chronic pain animal models . These changes include synaptic and intrinsic plasticity. We reported that increased spike number in response to current injection and reduced rheobase in the CeA-SST neurons in the MP mice , whereas their input resistance and resting potential were unaltered . Therefore, we speculate that some active conductances such as voltage-gated sodium channels are upregulated in the CeA-SST neurons of the MP mice. It is worth noting that increased excitability was specific to early-spiking (ES) neurons, the subtype of CeA-SST neurons in the MP mice. Conversely, the decreased excitability was specific to the ES type of PKCδ neurons. According to previous studies, modifications of intrinsic excitability are likely dependent on the PKC-ERK pathway . We discussed the possible mechanisms in Discussion .References:4, 394–396. Doi: 10.4161/cib.4.4.15694.Min MY, Yang HW, Yen CT, Chen CC, Chen CC, Cheng SJ. (2011). ERK, synaptic plasticity and acid-induced muscle pain. Commun Integr Biol 95, 135–142. Doi: 10.1177/0022034515618382.Sun, WH, and Chen, CC. (2016). Roles of Proton-Sensing Receptors in the Transition from Acute to Chronic Pain. J Dent Res 31, 2258–2270. Doi: 10.1523/JNEUROSCI.5564-10.2011.Cheng SJ, Chen CC, Yang HW, Chang YT, Bai SW, Chen CC, Yen CT, Min MY. (2011). Role of extracellular signal-regulated kinase in synaptic transmission and plasticity of a nociceptive input on capsular central amygdaloid neurons in normal and acid-induced muscle pain mice. J Neurosci 4, 26. Doi: 10.1186/1744-8069-4-26.Fu Y, Han J, Ishola T, Scerbo M, Adwanikar H, Ramsey C, Neugebauer V. (2008). PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior. Mol Pain 3) The Authors reported effects on amplitude and/or frequency of sEPSCs and mEPSCs onto CeA recorded neurons seem to suggest a presynaptic mechanism of action. In contrast, the effect of PGB on calcium transient revealed by 1P deep calcium imaging may be interpreted as pre and/or postsynaptic.The Authors should make a more definitive conclusion whether they think their data suggest a presynaptic only or also a postsynaptic mechanism of action. It may be difficult to draw any conclusion from sEPSCs since these events are probably due to the release of multiple synaptic vesicles .Our data showed that PGB suppressed synaptic transmission and increased the PPR at the PBN-CeA-SST neuron synapses . Therefore, we interpreted the suppression of calcium transients in the CeA-SST neurons by PGB was mediated by presynaptic inhibition .Regarding the mechanism of synaptic plasticity, we reported that both the sEPSC and mEPSC frequency were increased in the CeA-SST neurons. Therefore, the enhancement of vesicle release at the inputs onto the CeA-SST neuron synapses along with the increased excitability might mainly account for the central sensitization of the CeA-SST neurons in MP .4) The Authors should discuss the possible source(s) of the spontaneous synaptic events. Do they only originate from PBN terminals or also from BLA principal neurons or elsewhere? Is an analysis of mEPSCs kinetics compatible with an homogeneous pool of presynaptic terminals as source of the m/sEPSCs or rather SST+ and PKCδ+ neurons are biophysically too compact to make out the presence of heterogeneous excitatory inputs perhaps impinging on different spatial domains of the neurons' dendritic trees?In revision, we discussed the possible sources of the spontaneous synaptic events . Several excitatory inputs impinge onto CeA neurons . The CeA receives nociceptive inputs from the dorsal horn via the PBN and affect-related information from the LA . The PBN-CeA synaptic potentiation is consolidated in chronic neuropathic pain . In addition, BLA-CeA synapses are potentiated in chronic neuropathic pain . The PVT, which is a structure that is readily activated by both physical and psychological stressors, also projects to the CeA . Taken together, our results revealed that the net synaptic efficacy of the PBN-CeA, PVT-CeA or BLA-CeA synapses onto CeA-SST neurons were strengthened, whereas the synapses onto CeA-PKCδ neurons were weakened in chronic muscle pain.Our preliminary analysis of sEPSC decay time revealed at least two populations of sEPSCs, suggesting heterogeneous excitatory inputs impinging on different spatial domain of the neuron’s dendrites .References:49, 141–147. Doi: 10.1016/j.conb.2018.02.009.Fadok JP, Markovic M, Tovote P, Lüthi A. (2018). New perspectives on central amygdala function. Curr Opin Neurobiol 161, 166–176. Doi: 10.1097/j.pain.0000000000001691.Li JN, and Sheets PL. (2020). Spared nerve injury differentially alters parabrachial monosynaptic excitatory inputs to molecularly specific neurons in distinct subregions of the central amygdala. Pain 29, 332–346.e5. Doi: 10.1016/j.celrep.2019.09.011.Wilson TD, Valdivia S, Khan A, Ahn HS, Adke AP, Martinez Gonzalez S, Sugimura YK, Carrasquillo Y. (2019). Dual and opposing functions of the central amygdala in the modulation of pain. Cell Rep 127, 161–172. Doi: 10.1016/j.pain.2006.09.003.Ikeda R, Takahashi Y, Inoue K, Kato F. (2007). NMDA receptor-independent synaptic plasticity in the central amygdala in the rat model of neuropathic pain. Pain 519, 455–459. Doi: 10.1038/nature13978.Penzo MA, Robert V, Tucciarone J, De Bundel D, Wang M, Van Aelst L, Darvas M, Parada LF, Palmiter RD, He M, Huang ZJ, Li B. (2015). The paraventricular thalamus controls a central amygdala fear circuit. Nature 5) The paw withdraw effect could be replicated by either reducing the CeA SST+ cell activity or by elevating PKCd+ cell activity, but this is not the case for the anxiety-like affective behaviors . Therefore, the PGB effect on the increased paw withdraw threshold and emotion behaviors in the MP mouse model may be caused by distinct circuit mechanisms occurring simultaneously.To further elucidate their observation, the Author should test the acute PGB effect on the mechano-sensitivity and emotional behavior separately using a wild type mice or should at least acknowledge this possibility in the discussion.Distinct circuit mechanisms could account for the increased paw withdraw threshold and improved emotion behaviors in the MP mouse model by PGB because PGB treatment is not specific to the inputs onto the CeA-SST neurons. Nevertheless, our Figure 2 —figure supplement 1 provides a parsimonious explanation for the effect of PGB on the increased paw withdraw threshold and emotion behaviors.Regarding the PGB effect on mechano-sensitivity and emotional behavior, we plotted the changes of immobility time in the FST or number of buried marbles in the marble burying test against the change of mechano-sensitivity. Only the data obtained from the FST and marble burying test were used to address this question because these two behavioral assays could be tested before and after PGB application on the same animals. After PGB application, no positive correlation was observed between the change of the PW threshold and the change of the immobility time. A weak positive correlation between the change of PW threshold and the change in the number of buried marbles , suggests that PGB alleviates the mechano-sensitivity and emotional behavior through distinct circuit mechanisms . For the EPM and social avoidance, the results can be influenced by repeated tests. Therefore, animals were only tested with either ACSF or PGB.Reviewer #2 (Recommendations for the authors):My specific issues are outlined below:1) Why does the number of animals tested in the different behavioural paradigms  vary from test to test? Were these different cohorts for each behavioural test?We indeed had different cohorts of mice for each behavioral test. This is somehow inevitable during the development of a project. For the same cohort, we randomly and equally assigned animals to the MP and Ctrl groups and then performed behavioral tests. Initially we focused on some common anxiety-like behavioral tests such as the EPM, L/D box, and marble burying tests. After obtaining positive results, we decided to add additional behavioral tests to strengthen the face validity of this pain model. Anxiety disorders are often associated with depression-like behavior and social avoidance. Therefore, we added the FST for depression and the three-chamber test for sociability. Addition of different cohorts for each behavioral test accounts for different numbers of animals in the different paradigms.2) The size of the cohorts in which PGB was tested (and the relative controls) after induction of chronic muscle pain  is suboptimal for the EPM and sociability tests, also in view of the known relatively high interindividual variability, and should, therefore, be increased.We agree with the reviewer that the size of the cohorts is suboptimal for the EPM and sociability tests . In revision, we increased the number of experiments in Figure 2B  and 2E . The following is the summary of the results.p = 0.052. MP: ACSF, 15.1 ± 3.0 %, n = 7; PGB, 38.4 ± 5.1 %, n = 7; Mann–Whitney test, U = 2, **p = 0.002; Figure 2B) and increased the social-zone time in the three-chamber social test . PGB at the dosage used in this study had no effect on the locomotion .Compared to the Ctrl mice, cannula infusion of PGB into the CeA of the MP mice increased the open-arm time in the EPM test  It should be made more clear in the text that in vivo caCompared to the MP group, PGB had little effect on the mechanical sensitivity  and affective behaviors  in the Ctrl mice. Our finding suggests that PGB preferentially exerted its effect on the rewired pain/emotional circuits.We do agree that the reviewer raised an important question. Compared to the rewired circuitry in the MP mice, the effect of PGB on neuronal activity in the Ctrl mice could be different. Considering that PGB had little effect on pain behavior in the Ctrl mice, we speculate that PGB could exert a marginal effect on SST neuron activity in the Ctrl mice. Nevertheless, we agree with the reviewer that testing the effect of PGB on SST+ neuron activity in the control mice would strengthen our findings and should be addressed in the future.2+ transients. In this study, we focused on maladaptive changes of pro-nociceptive neurons  during the development chronic muscle pain. Therefore, we did not test the nociceptive response of the SST+ neurons in response to acute nociceptive stimulation such as acid injection.Regarding testing the effect of PGB on SST+ neuron activity during a von Frey filament stimulation, we agree that this experiment is interesting. Also, it may offer good behavioral bouts for detecting mechanically evoked caReviewer #3 (Recommendations for the authors):I have the following comments, which require more experiments to address in order to make the manuscript more convincing.1, Figure 3 shows recording results from CeA-SST and CeA-PKCd neurons in the MP mice. Since these neurons at baseline consist of different firing phenotypes , it would be useful to know whether all firing types show the same changes in synaptic transmission and excitability in the MP model.Notably, in Wilson et al., 2019 paper, they found that CeA neurons require at least 50-80 pA current injection to induce the spikes, whereas in this manuscript, CeA neurons require a much lower current injection to fire. Could the author provide some explanation to this difference?According to our previous study , we analyzed all CeA neurons shown in Figure 3 and classified them into early-spiking (ES) and late-spiking (LS) neurons based on their spike delay at rheobase. Using the same criteria, the CeA neurons with a spike delay greater than 1.5 s were classified as LS neurons, while the rest were classified as ES neurons . The ratio of these two subpopulations (ES vs. LS type) was not significantly altered in the MP mice.p = 0.006). However, the sEPSC amplitude recorded from SST-ES neurons in the MP mice was not significantly different from that in Ctrl mice . In contrast, the mean frequency  and amplitude  of SST-LS neurons in the MP mice were similar to those in the Ctrl mice.For the changes in excitatory synaptic transmission , compared to the Ctrl mice, sEPSC frequency was increased in SST-ES neurons of the MP mice . Similarly, the sEPSC amplitude in PKCδ-ES neurons of the MP mice was not significantly different from that in the Ctrl mice . In contrast, sEPSC frequency in PKCδ-LS neurons of the MP mice was significantly decreased compared to the Ctrl mice . However, sEPSC amplitude in PKCδ-LS neurons of the MP mice was unchanged compared to the Ctrl mice . Taken together, the upregulated excitatory transmission was specific to SST-ES neurons, whereas downregulated excitatory transmission was specific to PKCδ-LS neurons.On the other hand, sEPSC frequency in PKCδ-ES neurons of the MP mice was unchanged compared to the Ctrl mice . In agreement with increased excitability, the rheobase of SST-ES neurons in the MP mice was significantly lower than those in the Ctrl mice . Intriguingly, the number of evoked spikes  = 1.618, p = 0.206) and rheobase of SST-LS neurons  in the MP mice were similar to those in the Ctrl mice.For the changes in cell excitability , the SST-ES neurons in the MP mice generated more action potentials in response to depolarizing current injections compared to those in the Ctrl mice  = 10.84, **p = 0.0002). However, the rheobase of PKCδ-ES neurons in the MP mice was comparable to those in the Ctrl mice . Similar to SST-LS neurons, the spike number  = 2.84, p = 0.096) and rheobase  of PKCδ-LS neurons in the MP mice were similar to those in the Ctrl mice. Taken together, the modification of cell excitability in the MP mice is specific to ES neurons in both CeA-SST and CeA-PKCδ neurons.In great contrast to SST-ES neurons in the MP mice, the PKCδ-ES neurons in the MP mice generated less action potentials in response to depolarizing current injections compared to those in the Ctrl mice  = 16.4, ***Regarding the difference in current threshold for spike generation, the rheobase was measured in the presence of synaptic GABAA (1 μM) and GABAB (1 μM) receptor blockers in our study. In contrast, spikes were evoked in the absence of synaptic blockers in the study by Wilson et al. (2019). Therefore, reduced inhibition in our recordings could account for the requirement of a much lower current injection for spike generation.References:36, 4549–4563. Doi: 10.1523/JNEUROSCI.3309-15.2016.Hou WH, Kuo N, Fang GW, Huang HS, Wu KP, Zimmer A, Cheng JK, Lien CC. (2016). Wiring specificity and synaptic diversity in the mouse lateral central amygdala. J Neurosci 29, 332–346.e5. Doi: 10.1016/j.celrep.2019.09.011.Wilson TD, Valdivia S, Khan A, Ahn HS, Adke AP, Martinez Gonzalez S, Sugimura YK, Carrasquillo Y. (2019). Dual and opposing functions of the central amygdala in the modulation of pain. Cell Rep 2, The number of animals in each test is quite different. For instance, in Figure 4E, n=10 for the mCherry group and n=52 for the hM4Di group; this is a big difference to compare 2 groups. Also, in Figure 4 supplement 2, in the CeA-PKCd experiment, n=4 for the mCherry group and n=13 for the hM3q group. These numbers are significantly fewer than those for CeA-SST hM4Di group (n=52). Please explain how the numbers of animals were assigned to each test? Did the author exclude some animals from some tests?We designed two types of experiments to test the effect of silencing the CeA-SST neurons. Initially we tested the hM4Di effect on the MP mice . To this end, we expressed hM4Di on the SST neurons and then tested the effects of VEH  vs. CNO (i.p.) on the mechanical sensitivity in the MP mice. The result suggested that silencing the CeA-SST neurons by CNO reduces mechanical hypersensitivity. However, a potential caveat of this experiment is the off-target effect of CNO . Therefore, we performed another group of experiments, in which we tested the effect of CNO on the mCherry- and mCherry-hM4Di- expressing MP mice .In the originally submitted manuscript, we pooled the data obtained from the mCherry-hM4Di-expressing MP mice, which received the CNO treatment from the two experimental groups (n = 43 vs. 9). In revision, we separated the results of these two sets of experiment results. We only showed the CNO effect on the mCherry- and hM4Di-expressing MP mice  in Figure 4E. Since the result is sufficient to support our conclusion, we moved the data, which compared the CNO vs. vehicle on the hM4Di-expressing MP mice, to the supplemental part .References:8, 3840. Doi.org/10.1038/s41598-018-22116-z.Manvich DF, Webster KA, Foster SL, Farrell MS, Ritchie JC, Porter JH, Weinshenker D. (2018). The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice. Sci Rep 3, The authors did not show any post-hoc images of the virus injection/expression and co-localization with cell type specific markers for all the functional manipulation and imaging studies. These should be provided to substantiate the results.+ cells in the CeA are SST immunoreactive. Conversely, 63.63 ± 3.83 % SST-immunoreactive cells in the CeA are mCherry+ cells .In revision, we added images of virus expression (AAV5–hSyn-hM4Di-mCherry or AAV5–hSyn-hM3Dq-mCherry) colocalized with cell type specific markers (SST or PKCδ) in Figure 4B and Figure 4 —figure supplement 1A, 4B in the revised submission. The colocalization of specific markers with virus expression was summarized in Figure 4 —figure supplement 1B and 4B in the revised submission. Briefly, we injected an AAV5–hsyn-hM4Di-mCherry virus into the CeA of SST-Cre mice and performed SST immunohistochemistry (IHC) after virus expression . Our results revealed that 79.68 ± 1.87 % mCherry+ cells in the CeA are PKCδ immunoreactive. Conversely, 34.11 ± 1.86 % PKCδ-immunoreactive cells in the CeA are the mCherry+ cells .On the other hand, we injected an AAV5–hsyn-hM3Dq-mCherry virus into the CeA of PKCδ-Cre mice and performed PKCδ IHC after virus expression . Our results revealed that 62.73 ± 2.09 % mCherry4, In Figure 4 supplement 1B: Chemogenetic inhibition of CeA-SST neurons does not affect the paw withdrawal response in the naïve mice. In contrast, the authors themselves showed in their recent paper that optogenetic inhibition of CeA-SST neurons increased paw withdrawal response . Please reconcile these two different results in the same lab.-, which could alter the EGABA and reverse the GABAergic action . In our study, CeA-SST neurons receive reciprocal inhibition from neighboring CeA neurons. In our recent paper  and our unpublished data, yellow-light stimulation was applied to the naïve mice to activate the NpHR3.0 for 10 minutes. This could lead to intracellular Cl- accumulation after termination of light pulse and cause an increase in excitability or rebound action potential generation in the CeA-SST neurons. Because of several experimental parameters of optogenetic manipulations can result in different behaviors, we decided to employ chemogenetic manipulations in this study.The reviewer pointed out a key issue regarding the use of in vivo optogenetic manipulations and interpretation of animal behavior. Indeed, we also performed optogenetic inhibition (5-min illumination) of CeA-SST neurons in the control mice in this study (data not shown in the submitted manuscript). The result showed that the mechanical sensitivity was reduced. It was different from our recent paper  that optogenetic inhibition (10-min illumination) of CeA-SST neurons increased mechanical sensitivity . It seems that different illumination protocols resulted in opposite behaviors. Several factors could confound interpretation of optogenetic experiments. First, illumination protocols and intracranial heating could activate or inactivate temperature-sensitive ion channels and thus bias animal behavior . Second, long-term optogenetic inhibition is known to cause accumulation of intracellular CIReferences:22, 1061-1065. Doi: 10.1038/s41593-019-0422-3.Owen SF, Liu MH, Kreitzer AC. (2019) Thermal constraints on in vivo optogenetic manipulations. Nat Neurosci 15, 1102-1104. Doi:10.1038/nn.3143.Raimondo JV, Kay L, Ellender TJ, Akerman CJ. (2012). Optogenetic silencing strategies differ in their effects on inhibitory synaptic transmission. Nat Neurosci 163, e463-e475. Doi:10.1097/j.pain.0000000000002389.Chen WH, Lien CC, Chen CC. (2022). Neuronal basis for pain-like and anxiety-like behaviors in the central nucleus of the amygdala. Pain 5, There is no data on a2d VGCC subunit expression in parabrachial or CeA in the chronic MP model in this manuscript. Given so much emphasis on PGB results, this information is crucial and is missing here.Thanks for the reviewer’s comments. Cole et al., 2005 had described the anatomical distribution of α2δ VGCC subunit expression across the whole rat brain. It revealed that the α2δ VGCC subunit is expressed in the parabrachial nucleus and CeA. In the revised submission, we also provide published data by Allen Brain Institute , which show α2δ1 and α2δ2 VGCC subunit expression in the parabrachial in our supplement file .References:491, 246-269. Doi:10.1002/cne.20693.Cole RL, Lechner SM, Williams ME, Prodanovich P, Bleicher L, Varney MA, Gu G. (2005). Differential distribution of voltage-gated calcium channel α-2 δ  subunit mRNA-containing cells in the rat central nervous system and the dorsal root ganglia. J Comp Neurol 6, The authors performed the in vivo calcium imaging of CeA-SST neurons in response to PGB. It would be more informative to know how CeA-SST neurons or CeA-PKCd neurons respond to 1st and 2nd acid injection.st and 2nd acid injection. However, it is not the focus of this study. We speculate that several induction protocols including acidic saline injection or complete Freund’s adjuvant injection for chronic pain can differentially activate these two CeA neurons subtype. The latency of their activation in response to induction stimuli may be different. We plan to investigate how CeA-SST neurons or CeA-PKCδ neurons activate during induction in different types of chronic pain mouse models in the future.Indeed, it would be interesting to know how CeA-SST neurons or CeA-PKCδ neurons respond to 1Specifically:1) The text is often hard to read and should be carefully checked to improve readability. If needed, a professional writer could be helpful.To improve the readability of the manuscript, we have submitted the manuscript to the Wiley Editing Services for professional editing.2) All the reviewers acknowledged the additional excel database and your explanations regarding the data in Figure 3 – supplement 2 B-D. These data were submitted to support the electrophysiological results – that SST+ neuron excitability is selectively enhanced in MP mice. However, some improvements are needed:a) We think that you pooled the data from "all slices" together at a given AP position relative to Bregma, to generate the plots and calculate SEM. However, we think you should re-calculate the mean at each AP position for each animal first, then average across animals and calculate SEM. In this way, the data would be averaged within an animal and the variance would be calculated between animals in order to assess the robustness of the results;p = 0.016; Figure 3 —figure supplement 2B, right). Notably, the total number of SST(+)pERK(+) in the CeA was increased in the MP mice . In contrast, the total number of SST(-)pERK(+)cells in the CeA was slightly, but not significantly, increased in the MP mice .In the revised version of the manuscript, we conducted a new set of experiment . As the reviewer suggested, we calculated the mean at each AP position for each animal first, then average across animals and calculate SEM . Similar to our previous results, the number of pERK cells was increased in the MP mice as compared to the Ctrl mice  Explain why the number of slices at a given AP position varies dramatically across animals . Furthermore, how it could be that in some mice the AP position has 5-9 slices, whereas from the method one can calculate that a maximum of 3 slices can be obtained for a given AP position;Thanks for pointing out this potential problem. The reason why some animals have 1 slice and some have 5-9 slices at a given AP position is because of the variation in the slice integrity and the slicing angle of each mouse. We agree with the reviewer that the number of slices should be consistent at a given AP position in an animal and should be less variable across animals. To this end, we conducted a new set of experiment and collected 2-5 slices at each AP position per mouse.c)Tthe number of samples for each bregma level and animal should be similar and more control data should be collected and included since currently they show large variability;In the revised version, we conducted a new set of experiment  to have the same number of animals in each group.d) Statistical analysis should be improved, e.g. a two-factor analysis of variance  would be a good way to analyze these data.p <0.0001 ; Figure 3 —figure supplement 2B, left). Similarly, the number of SST(+)pERK cells was increased in the MP mice as compared to the Ctrl mice  = 34.12; p <0.0001 ; Figure 3 —figure supplement 2C, left). Moreover, the numbers of SST(+)pERK(+) neurons at -1.46 and -1.58 AP position in the MP mice were significantly greater than those in the Ctrl mice  = 34.12, *p = 0.039; AP: -1.58: Ctrl, 0.68 ± 0.49, n = 5; MP, 3.06 ± 0.61, n = 5; two-way ANOVA with Bonferroni's multiple comparison test, F = 34.12, *p = 0.047; Figure 3 —figure supplement 2C, left). Likewise, the number of SST(-)pERK(+) cells in the CeA along the AP axis was increased in the MP mice as compared to the Ctrl mice  = 24.4; p <0.0001 ; Figure 3 —figure supplement 2D, left).In the revised version, we conducted a new set of experiment. Both control and MP groups had equal numbers of animals. As the reviewer suggested, we used the two-way ANOVA with the Bonferroni's multiple comparison test to calculate the statistic power in the diary plot of panel B-D. Consistent to our previous results, the total number of pERK cells in the CeA along the AP axis was increased in the MP mice as compared to the Ctrl mice  = 29.66; p = 0.016; Figure 3 —figure supplement 2B). Similarly, the total number of SST(+)pERK(+) cells in the CeA was also increased in the MP mice . In contrast, the total number of SST(-)pERK(+) cells was slightly, but not significantly, increased in the MP mice .Finally, we took the comment of the reviewer 3 and compared the total number of cells in the CeA, instead of the averaged number across sections. Compared to the Ctrl mice, the total number of pERK cells was increased in the MP mice :I think that the Authors have done a good job in revising the manuscript according to my suggestions.Thanks for the positive comment of the reviewer 1.Reviewer #2 (Recommendations for the authors):The work by Lin and coworkers addresses the important and of broad interest process that underlies the progression of transient pain into persistent pain. This study used a number of sophisticated techniques to dissect the synaptic and cellular mechanisms that occur in the mouse central amygdala (CeA) during the development of chronic muscle pain. In particular, the study reveals shifts in opposite directions of the activity of somatostatin-expressing (SST+) and PKCδ+ neurons. Pharmacological and chemogenetic manipulation of these neurons prevented the chronification of pain and of the comorbid affective behaviors, although with important differences between these two neuronal classes.In their revised manuscript, the authors have made a number of significant changes by which they could adequately address my main concerns. In particular, the additional experimental data aimed at increasing the sample size of the behavioural experiments has appreciably improved the manuscript.While the text has improved, it still contains several syntax and grammar errors that need to be corrected.Thanks to the positive comment of the reviewer 2. We have rechecked the manuscript and corrected the syntax and grammar errors. Furthermore, we have submitted the manuscript to the Wiley Editing Services for improvement of readability.Reviewer #3 (Recommendations for the authors):I do have some questions for Figure 3—figure supplement 2+ cells in B being less than that in D (a subset of neurons)?In D, the number of SST(-)pERK(+) in the Ctrl (-1.82 mm) is above 40, while in B, the number of total pERK(+) in the Ctrl (-1.82mm) is about 30. This is weird. How could total number of pERKIn the previous version, we found that we mistakenly caused a slight shift of the control diary plot in the panel B. Together with the reviewer’s concern about the variability of slices at a given AP position and the different number of animals between the control and MP groups, we discarded the previous data and conducted a new set of experiment .Authors concluded in Line 212-213, "increased pERK-positive cells are mostly CeA-SST cells." From the results shown in C and D, it's difficult to be convinced of this statement: the number of SST(+) pERK(+) cells increased from 1.1 to 3.7, while SST(-) pERK(+) cells increased from 18 to 28, even though statistics appeared to be not significant. I believe that instead of comparing the averaged number across sections, comparing the total number of cells in the CeA makes more sense.p = 0.016; Figure 3 —figure supplement 2B). Similarly, the total number of SST(+)pERK(+) cells in the CeA was also increased in the MP mice . In contrast, the total number of SST(-)pERK(+) cells was slightly, but not significantly, increased in the MP mice .For the new data, we took the reviewer’s comment and compared the total number of cells in the CeA. Compared to the Ctrl mice, the total number of pERK cells was increased in the MP mice  and in panel D (SST-/pERK+) appear identical. Could there be an image duplication here?The "gray" bars for "control" samples in panel B  and in panel D (SST-/pERK+) appear similar, but not identical. Consistent with our old result, the diary plots and histograms in the new data indeed showed similar distribution .In our old data, the "gray" bars for "control" samples in panel B (total pERK


Moreover, specific antibody levels can be restored after the boosting vaccination measured by ELISA. Furthermore, the level of SARS-CoV-2 specific IgG response is independent of age and gender. Moreover, N391-408 was identified as a dominant peptide after vaccination of BBIBP-CorV through peptide screening. Understanding the overview of humoral reactivity of the vaccine will contribute to further research on the protective mechanism of the SARS-CoV-2 inactivated vaccine and provide potential biomarkers for the related application of inactivated vaccine.Vaccination is one of the best ways to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic. Among the various SARS-CoV-2 vaccines approved for use, the BBIBP-CorV inactivated vaccine has been widely used in 93 countries. In order to understand deeply the protective mechanism of inactivated vaccine, which retains all antigenic components of live virus, the analysis of humoral responses triggered by multiple proteins is necessary. In this research, antibody responses were generated with 6 selected recombinant proteins and 68 overlapping peptides that completely covered SARS-CoV-2 nucleocapsid (N) protein in 254 healthy volunteers vaccinated with BBIBP-CorV. As a result, antibody responses to the receptor binding domain (RBD), N, and non-structural protein 8 (NSP8) were induced following immunization by BBIBP-CorV. The antibody responses detected in donors after the 2 So far On a global scale, herd immunity through vaccination is the best way to return to normal life . AccordiThe BBIBP-CorV is the most widely used inactivated SARS-CoV-2 vaccine in the world, which has been approved in 93 countries. The SARS-CoV-2 strain of BBIBP-CorV  was isolated from the bronchoalveolar lavage sample of a hospitalized patient at the beginning of the outbreak. The BBIBP-CorV was inactivated by β-propionolactone treatment and prepared with aluminum hydroxide as adjuvant . A randoThe immune responses of SARS-CoV-2 have been well studied in infected and vaccinated individuals. At present, the humoral immunity research of inactivated vaccines mainly focus on five aspects: immunogenicity and neutralizing activity , neutralThis study was conducted to understand the IgG and IgM responses triggered by inactivated SARS-CoV-2 vaccine, BBIBP-CorV. Serum samples from 254 healthy individuals vaccinated with the homologous BBIBP-CorV inactivated vaccine were analyzed. Two structural proteins  and four non-structural proteins  were initially selected for antibody response analysis. We chose RBD because its antibody titer is correlated with antibody neutralization ability . The N pThis study was approved by the Ethics Review Committee of the Eighth Affiliated Hospital of Sun Yat-sen University (reference 2021-005-01). Under the principles of the Helsinki declaration, the written informed consent of the participants was obtained.Serums were collected from 254 volunteers who had been examined at the Eighth Affiliated Hospital of Sun Yat-sen University since July 2022. After coagulation, serum was collected in an IMPROVE Vacutainer SST tube. The serum was centrifuged at 3,500 rpm for 10 min and was separated before preservation at −80°C. The demographic and clinical characteristics of the volunteers were shown in E. coli BL-21. After verification by sequencing, induction expression of recombinant protein was carried out. E. coli (1:100) was added to LB medium (Kana-resistance) and was shaken until OD600nm was 0.8. IPTG  was added and continued culture for 3 h. It’s confirmed by electrophoresis that proteins expressed in the supernatant after ultrasound. Then, the ultrasonic supernatant was purified by AKTA pure (GE) system. The ultrasonic supernatant was transferred to Ni-NTA prepacked chromatographic column . The eluent was a Tris–HCl buffer solution containing 250 mM imidazole. Then an ultrafiltration tube  was used for eluent concentration and buffer replacement. Protein purity was determined by SDS-PAGE electrophoresis. The concentration of proteins was determined by BCA . N, NSP1, NSP5, NSP7, and NSP8 proteins used in this study were prepared in our laboratory  to construct expression plasmids using PET-28a as a vector and transferred into tory see  and RBD The NCBI entry number used to design the SARS-CoV-2 nucleocapsid linear peptide sequence is YP_009724397.2. The principle of overlapping peptides design: offset six amino acids and overlap 12 amino acids. The N protein peptide library  consists of sixty-eight 18-mer overlapping peptides see , which cAn indirect ELISA assay for the detection of IgG and IgM responses of selected proteins and peptides was performed according to the following instructions. In brief, ELISA plates  were coated with the recombinant proteins (a 2 μg/l carbonate solution) or peptides  at 4°C overnight. ELISA plates were blocked with 1% BSA-PBST (0.05% Tween-20) for 2 h at 37°C after washing 3 times with PBST (0.05% Tween-20). 100 μl serum samples  were added to each well and incubate for 1 h at 37°C before washing 5 times. Next, 100 μl HRP labeled goat Anti-human IgG  or HRP labeled goat anti-human IgM  were used for detection of antibodies. In total, 100 μl TMB  was used for a 10 min development and was stopped by addition of 100 μl of stop solution , prior to absorbance measurements. OD450nm value was measured with the microplate reader (ThermoFisher).N protein sequences of seven human coronavirus-infected species (accession number: YP_009724397.2 AAP33707.1 YP_00904721.1 YP_009555244.5 1.1 YP_173242.1) YP_003771.1 and NP_073556.1) were downloaded from NCBI database. N protein sequences of Six VOC and four omicron variants were downloaded from the COVID-19 database.t-test, Mann–Whitney test, ordinary one-way analysis of variance (ANOVA), Kolmogorov–Smirnov test, and Pearson correlation were analyzed by GraphPad Prism 9.0. The hypothesis test was two-sided, and we considered the p-value <0.05 to be significant.A positive control has been selected out to account for plate-to-plate variations. Corrected OD450nm values of samples were normalized to the positive control, and background signals were subtracted. Signal Intensity was defined as the test value minus blank value in OD450nm. Two independent repeated experiments were performed for each sample. The dominant peptide was the one that tested the highest Corrected OD450nm value. Dominant Frequency (%) was calculated as dominant peptide (pool) frequency divided by total frequency. Student’s st, 2nd, and 3rd dose, respectively. For the 2nd dose group, volunteers were divided into five subgroups according to the time post vaccination. The demographic and clinical characteristics of the volunteers were summarized in 254 volunteers were recruited and serum samples were collected. Among them, 31 participants were unvaccinated. The left 31, 161 and 31 donors were vaccinated by BBIBP-CorV post the 1nd dose group. In terms of IgG response . The IgG and IgM antibody responses to different proteins were detected and compared between the unvaccinated group and the 10–30 days post the 2response –F, the Iresponse –L, the Iresponse  Notably,response –K. In gest and 2nd dose of vaccination, serum samples from the 1st dose immunization group, and 10–30 days post the 2nd dose of vaccination group were analyzed, with the IgG and IgM responses of unvaccinated group investigated as control. Specific IgG and IgM antibody levels for RBD, N, NSP8 among these three groups were detected via indirect ELISA assay. For IgG antibody , but no RBD IgG response was found (1.13-fold). After the 2nd dose of vaccination, although there was no statistically significant increase in N and NSP8 IgG levels compared with the 1st dose , but still showed increasing trends. Besides, some individual’s IgG against N protein have increased considerably. Meanwhile RBD-specific IgG antibody increased significantly (3.03-fold) after the 2nd dose vaccination. For specific IgM responses , which was further elevated by the second one . Overall, elevated N and NSP8 IgG antibody response were observed after the 1st dose, boosted antibody levels of anti-N, -NSP8 IgM, and anti-RBD IgG/IgM were observed post the 2nd dose vaccination. These results suggest that secondary vaccination strategy of BBIBP-CorV is necessary.To compare the change of antibody levels after the 1antibody –C, the 1esponses –F, the 1nd dose of vaccination. In this study, the RBD-, N-, and NSP8-specific IgG and IgM responses were detected by indirect ELISA method. The specific IgG and IgM responses against those 3 antigens showed the same trend as antibody levels increased to a plateau and then decreased over time after the 2nd dose vaccination of BBIBP-CorV. In specific IgG antibody response , which was equivalent to the degree of the 2nd dose group (2.07-fold) and showed an increasing trend. The laws of evolution for N-specific IgG response were the same as that of RBD. Three-dose of the BBIBP-CorV also boosted N-specific IgG response (2.60-fold) compared to the 2nd dose >90d group (1.51-fold). NSP8-specific IgG increased after the 3rd dose, but the difference was not statistically significant. For specific IgM response . We compared the sequences of N391-408 to that of all coronaviruses known to infect humans, the immune dominant N391-408 were highly specific between species (except SARS-CoV). By comparing the conservation degree of N391-408 among the Variants of Concern (VOC), N391-408 was 100% conserved among different mutant strains. It’s worthy noting that N391-408 was also conserved among currently epidemic omicron variants . Thirteen individuals whose N-specific IgG or IgM responses level are above cut-off value (x̄ + 3σ) were tested, respectively. These peptides were divided into eight peptide pools, each containing 8–9 peptides. The indirect ELISA assay was performed to detect peptide-specific IgG response . As a reN391-408 ,D. In suvariants . The spa protein  showed tVaccination is one of the best strategies to contain SARS-CoV-2 epidemic. Among the various SARS-CoV-2 vaccines approved for use, the BBIBP-CorV inactivated vaccine has been widely used in 93 countries. Nevertheless, the immune response of non-S protein after vaccination of inactivated vaccines is slightly less studied. This study was conducted to understand the IgG and IgM responses triggered by an inactivated vaccine, BBIBP-CorV, in a long term.At the protein level, results imply that SARS-CoV-2 specific IgG and IgM of RBD, N and NSP8 were induced by BBIBP-CorV in healthy populations compared with unvaccinated populations. Using inactivated vaccines have the characteristic of a broader immune response compared to other vaccine platforms that use only Spike protein. Strong antibody responses against S and N proteins also detected in COVID-19 patients . As a reWe believe that inactivated vaccines were able to induce protective antibodies, besides S-specific antibody. The N protein of CoVs has been shown to be involved in host cell mechanisms, such as immune escape interferon inhibition, RNA interference and apoptosis, and cytokine release . Moreovend dose, while N-, and NSP8-specific IgG mainly appears after the 1st dose, which indicates that the primary and secondary immunization can trigger different antibody response spectrums and worth further investigation. Because neutralizing antibodies primarily target RBD , was located in the C-tail region and set on the surface of N protein, which provided the foundation for its dominant response. To our knowledge, there is no study about the dominant epitope of nucleocapsid after vaccination of SARS-CoV-2 inactivated vaccines. A study based on infected individuals  and C-terminal domain (CTD) of SARS-CoV-2 N protein have been analyzed and the CTD owns a dominance compared with NTD . After sividuals  mapped tividuals , QKKQQTVividuals , AADLDDFividuals  and TVTLividuals . These e391-408 showed specificity among different viral species (except SARS-CoV) and is 100% conserved among mutant strains. Based on the protective immunity of N protein, we speculate that this epitope may be used as a complement to serological tests and vaccine efficacy evaluation. The localization information, that N391-408 is close to N-CTD, suggests that the role and mechanism of the N391-408 antibody after vaccination are worthy of further study.Antibody assay is a key test for assessing the effectiveness of SARS-CoV-2 vaccination, distinguishing vaccination from natural infection  and idenWe have to admit that there are several limitations. First, we failed to use paired samples for detection unfortunately. Instead, we tried to reduce the influence of individual differences by increasing the sample size. Second, the application of the dominant epitope needs further experimental verification. Finally, studies on the immunological function and mechanism of dominant epitope antibodies have not been included in this manuscript. Especially to deserve to be mentioned, another team in our group has started related research, and we believe that there will be new progress soon.In conclusion, by taking advantage of the SARS-CoV-2 protein-based ELISA and N protein peptide-based ELISA, we generated the SARS-CoV-2-specific IgG and IgM responses profile triggered by an inactivated SARS-CoV-2 vaccine, BBIBP-CorV. In this research we detected obvious differences by comparing this profile to those of unvaccinated volunteers. Furthermore, dynamics of SARS-CoV-2-specific antibody response after vaccination were identified and an immunodominant linear B cell epitope on the N protein that may have the potential to be used as a complement to serological detection and vaccine efficacy evaluation.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by the Ethics Review Committee of the Eighth Affiliated Hospital of Sun Yat-sen University (reference 2021-005-01). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.QW and CW drafted the manuscript. All authors edited and reviewed manuscript drafts. LS and AZ were responsible for recruiting volunteers and collecting human samples. FZ and XC provided recombinant proteins. QW and TH provided N protein peptide library. QW, JN, and BL performed immune assays. QW, JN, and YC were responsible for statistical analysis. All authors contributed to the article and approved the submitted version.This project was supported by Shenzhen Science and Technology Program (JCYJ20210324115204012) and Shenzhen Futian District Public Health Research Project (FTWS2020017).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


For the sake of the oxygen reduction reaction (ORR) catalytic performance, carbon dots (CDs) doped with metal atoms have accelerated their local electron flow for the past few years. However, the influence of CDs doped with metal atoms on binding sites and formation mechanisms is still uncertain. Herein, Co,N-doped CDs were facilely prepared by the low-temperature polymerization–solvent extraction strategy from EDTA-Co. The influence of Co doping on the catalytic performance of Co-CDs was explored, mainly in the following aspects: first, the pyridinic N atom content of Co-CDs significantly increased from 4.2 to 11.27 at% compared with the CDs, which indicates that the Co element in the precursor is advantageous in forming more pyridinic-N-active sites for boosting the ORR performance. Second, Co-CDs are uniformly distributed on the surface of carbon black (CB) to form Co-CDs@CB by the facile hydrothermal route, which can expose more active sites than the aggregation status. Third, the highest graphite N content of Co-CDs@CB was found, by limiting the current density of the catalyst towards the ORR. Composite nanomaterials formed by Co and CB are also used as air electrodes to manufacture high-performance zinc–air batteries. The battery has good cycle stability and realizes stable charges and discharges under different current densities. The outstanding catalytic activity of Co-CDs@CB is attributed to the Co,N synergistic effect induced by Co doping, which pioneer a new metal doping mechanism for gaining high-performance electrocatalysts. The serious energy crisis has been increasingly prominent in recent years, which promotes us to exploit new types of energy storage and conversion equipment ,2,3. For3 atmosphere , in a tube furnace at 350 °C for 2 h at a heating rate of 5 °C min−1 under a N2 atmosphere. Through the one-step calcination method, we can obtain carbon dots containing Co, named Co-CDs. When the calcination temperature is lower than 250 °C, the color of the carbonized product is fuchsia, the same as the precursor, which indicates that the synthesis condition makes it hard to carbonize the precursor to form Co-CDs. Increasing the temperature ranging from 250 °C to 350 °C, their color changes from fuchsia to black, demonstrating the construction of Co-CDs [The fabrication of the Co-CDs@CB catalysts was obtained by a two-step hydrothermal synthetic strategy, as depicted schematically in f Co-CDs . We can Although the calcination temperature is as high as 350 °C, the obtained Co-CDs exhibit a lowly graphitized structure, as shown in the XRD pattern . The pheThe morphology of the as-prepared catalysts of the CDs, Co-CDs, and Co-CDs@CB were characterized by transmission electron microscopy (TEM). It can be found that the CDs and Co-CDs have good dispersion on the ultra-thin copper grid. The average size of CDs is 4.5 nm a, which 2+ in CDs, and the doping of Co can improve the performances of CDs in the spectral and electrical properties [The chemical bonds, element composition, and state of the catalysts were detected by Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). In the full spectrum of X-ray photoelectron spectroscopy in operties . Additio−1, respectively. Compared to the Co-CDs, the CDs show an extra C-OH-stretching vibration peak at 1292 cm−1, which may be due to the incomplete carbonization of the precursor that leads to the residue. Meanwhile, we can find five characteristic absorption peaks for Co-CDs@CB in −1 are related to the O-H, N-H, C=O, C=C, and C-O-stretching vibration peaks, respectively. It is necessary to point out that the C-N bond is dispersed during the compounding process, which can be attributed to the Co-doped one and can be inferred that the doped atoms are most likely to combine with the CDs at the edge.As shown in onset), highest halfwave potential (E1/2), and highest limiting current density (jL). Specifically, the Eonset (0.9895 V) and E1/2 (0.8430 V) of Co-CDs@CB were significantly superior to those for the CDs (0.7384 and 0.4653 V), Co-CDs (0.7848 and 0.5945 V), CB (0.7089 and 0.5454 V), and CDs@CB (0.95 and 0.85 V). Compared with the Eonset (0.9962 V) and E1/2 (0.8657 V) of commercial PtC, it also has a strong competitive advantage. These results demonstrate that the introduction of Co-doping into the Co-CDs@CB electrocatalyst can provide vital contributions in gaining a higher ORR activity [L of 4.09 mA/cm2, which is superior to CDs (2.45 mA/cm2), Co-CDs (2.02 mA/cm2), CB (2.12 mA/cm2), and CDs@CB (0.95 mA/cm2), reflecting the outstanding mass transfer performance of Co,N-coordinated Co-CDs@CB architectures. Moreover, the excellent ORR activity of Co-CDs@CB can be further evidenced by the lowest Tafel slope (56 mV dec−1) (Cyclic voltammetry (CV) and Linear sweep voltammetry (LSV) were measured to access the electrocatalytic ORR activities of the as-prepared materials. activity . MeanwhiV dec−1) c, illustL increases with the promotion of the rotating speed. Meanwhile, the number of transferred electrons is 3.88–3.90, which conforms to the first-order-dynamic kinetics [2O2 (%) of Co-CDs@CB  plots, the assessment standard of catalytic selectivity. As shown in kinetics ,53. It io-CDs@CB f. The re−1 at 10 mA cm−2 , corresponding to an energy density of 711 Wh kg−1. The charge and discharge voltage cycles at the current density range from 10 to 100 mA cm−2, shown in −2. It is worth noting that, after more than 180 h of cycling, the voltage increase of the battery is very small, only 50 mV. According to the charge–discharge cycles at the current density of 10 mA cm−2 in Owing to the superior ORR reaction kinetics, the Co-CDs@CB was used as a catalyst to build a liquid zinc–air battery (ZAB), in which the foam nickel–carbon paper composite with Co-CDs@CB as the air cathode and a Zn sheet as the anode. As shown in p of 0.83 V, an Eonset of 0.99 V, an E1/2 half-wave potential of 0.84 V, and a limiting current density of 4.09 mA/cm2, respectively. Meanwhile, a complete four-electron ORR pathway was revealed by the K–L equation and RRDE measurements. The great enhancement in the ORR performance of Co-CDs@CB primarily lies in the synergy of the Co-CDs and CB, where the Co-CDs could provide abundant active edges/sites for electrocatalytic reactions, and CB could act as a conductive substrate to facilitate the electron transfer and also protect the Co-CDs from agglomeration to maximize the utilization efficiency of each active site. Through the measurement of zinc–air battery, we found that Co-CDs@CB has excellent performance as an air electrode catalyst. Even at a high current density of 100 mA cm−2, it has a stable discharge voltage. This work not only provides a simple strategy for designing CDs but also opens novel horizons for applying CDs for energy conversion and storage.In summary, Co,N-doped CDs were facilely prepared by the low-temperature polymerization–solvent extraction strategy from EDTA-Co. Based on the morphology measurements from TEM, the Co element in the precursor plays a vital role in suppressing the growth of Co-CDs and generating abundant active edge sites. Furthermore, the pyridinic N atom content of the Co-CDs significantly increased from 4.2 to 11.27 at% compared with the CDs, which indicates that the Co element in the precursor is advantageous in forming more pyridinic-N-active sites for boosting the ORR performance. In addition, Co-CDs are uniformly distributed on the surface of the CB to form Co-CDs@CB by the facile hydrothermal route, which can expose more active sites than the aggregation status. It is more likely to be chemically bonded to the carbon matrix, which is conducive to the electron transfer between the Co-CDs and CB. Additionally, the highest graphite N content of Co-CDs@CB was found to enhance the limiting current density of the catalyst towards the ORR. The Co-CDs@CB serving as an outstanding ORR catalyst exhibited an E